Pulmonary arterial hypertension (PH) is a devastating disease of diverse etiology that contributes to high morbidity and mortality of patients with various lung and heart diseases. PH is characterized by vascular obstruction and variable presence of vasoconstriction, leading to a sustained increased pulmonary vascular resistance, vascular remodeling, right ventricular (RV) failure, and low cardiac output. Despite its profound clinical consequences, therapeutic advances over the past 25 years have modestly improved survival of PH patients. The combined use of available drugs to maximize the clinical benefit is an emerging strategy for the treatment of PH. Although failing RV is a common clinical problem in PH, currently RV-specific experimental therapies are not available. One of the major physiologic mechanisms of PH suggests endothelial dysfunction that causes diminished nitric oxide (NO) production and NO/cyclic guanosine 5'monophosphate (cGMP)- dependent vasorelaxation in the pulmonary circulation. Alternatively, pulmonary vascular cGMP levels can also be elevated via inhibition of phosphodiesterase type 5 (PDE5) to achieve a similar response. Since PDE5 expression has been shown to be increased in the lungs and RV of PH patients, targeted efforts to increase NO production and PDE5 inhibition can counteract vasoconstriction that contributes to increased afterload in failing heart of PH patients. We have identified a fifteen amino acid (FKSRNSRWSCRIKSR) synthetic peptide (P3) that enhances the catalytic activity of endothelial NO synthase (eNOS) and inhibits cGMP-specific PDE5 in pulmonary artery endothelial cells as well as in pulmonary artery smooth muscle cells. Preliminary data indicate that P3 stimulation increases intracellular NO release, inhibits rate of cGMP hydrolysis, and attenuates increase in pulmonary artery pressure (PAP) but not systemic artery pressure (SAP) monitored in telemetry implanted conscious rat model of hypoxia-induced PH. Based on these preliminary data, we hypothesize that P3 treatment with its unique dual action as NO releasing PDE5 inhibitor offers novel therapeutic approach for the treatment of PH. To test this hypothesis, the specific aims of this study are to: (I) identify P3-mediated molecular events associated with increased eNOS activity and NO production with specific focus on Ca2+ release, activation of signaling modules [phospatidylinositide (PI) 3-kinase (PI3K), protein kinase B (PKB or Akt), protein kinase A (PKA), and Src tysorine kinase (SrcK)], phosphorylation of caveolin-1 (Cav-1) and/or eNOS, and Cav-1/eNOS dissociation, (II) elucidate whether P3 mediated inhibition of PDE5/cGMP hydrolysis is associated with Ca2+ release, activation of PI3K, protein kinase G (PKG)/PKA signaling, phosphorylation of PDE5, and/or inhibition of cGMP binding of PDE5, and (III) determine therapeutic potential of P3 using a physiologically-relevant hypoxia-induced animal model of PH with specific focus on assessment of bioavailability, dosing regimen, efficacy, and toxicity of P3 therapy on the progressive and regressive pulmonary hemodynamic modulations (increased PAP, SAP, cardiac output/cardiac index, hematocrit, and RV hypertrophy) and vascular remodeling associated with PH as well as toxicity. Confirmation of the mechanism-based physiological approach for NO releasing PDE5 inhibitor function of this novel peptide in preclinical animal model is innovative for progression towards Phase I clinical trial for treatment of PH. PUBLIC HEALTH RELEVANCE: Relevance to Veterans Health: Pulmonary vascular disease and hypertension are common in the VA patient population because of the high incident in this population of cigarette smoking-related chronic obstructive pulmonary disease (COPD). Vascular complications of pulmonary injury, including PH, are also frequently observed in combat casualty victims who develop adult respiratory distress syndrome (ARDS). In addition, exposure of veterans and current active duty personnel to multiple risk factors including high altitude, drugs, chemicals, HIV infection, and schistosomiasis can cause lung injury and progressive development of PH. Identification of mechanism-based unique therapeutic agent with NO releasing PDE5 inhibitory function offers a novel strategy for treatment of PH.